Li et al. Standards in Genomic Sciences (2017) 12:28 DOI 10.1186/s40793-017-0240-8

SHORT GENOME REPORT Open Access Complete genome sequence of Kosakonia oryzae type strain Ola 51T Yuanyuan Li1†, Shuying Li1†, Mingyue Chen1, Guixiang Peng2, Zhiyuan Tan3* and Qianli An1*

Abstract Strain Ola 51T (=LMG 24251T = CGMCC 1.7012T) is the type strain of the species Kosakonia oryzae and was isolated from surface-sterilized roots of the wild rice species Oryza latifolia grown in Guangdong, China. Here we summarize the features of the strain Ola 51T and describe its complete genome sequence. The genome contains one circular chromosome of 5,303,342 nucleotides with 54.01% GC content, 4773 protein-coding genes, 16 rRNA genes, 76 tRNA genes, 13 ncRNA genes, 48 pseudo genes, and 1 CRISPR array. Keywords: Endophyte, Kosakonia, Nitrogen fixation, Plant growth-promoting bacteria

Introduction the genetic information to study its plant growth- Enterobacter cowanii [1], E. radicincitans [2], E. oryzae promoting potential and its plant-associated life style. [3], E. arachidis [4], E. sacchari [5], E. oryziphilus [6, 7], and E. oryzendophyticus [6, 7] have been transferred into the novel genus Kosakonia of the family “Enterobacteria- Organism information ceae” [8–10]. A novel species “Kosakonia pseudosac- Classification and features chari” [11] closely related to K. sacchari was recently K. oryzae strain Ola 51T is a Gram-negative, non-spore- proposed. With the exception of the type species K. cow- forming, motile rod with peritrichous flagella (Fig. 1). It anii, which was originally obtained from clinical samples grows aerobically but reduces N2 to NH3 at a low pO2. [1], the other members of the genus Kosakonia are It forms circular, convex, smooth colonies with entire nitrogen-fixing bacteria associated with plants [2–6, 11] margins on nutrient agar [3, 8]. It grows best around and commonly occur in the nitrogen-fixing bacterial 30 °C and pH 7 (Table 1) [3]. K. oryzae Ola 51T has the community of some non-legume crops, such as rice [6] typical biochemical phenotypes of the genus Kosakonia: and sugarcane [12]. Some nitrogen-fixing Kosakonia positive for acetoin production (Voges-Proskauer test) strains are able to promote crop growth [12–14]. while negative for indole production; positive for β- Strain Ola 51T (=LMG 24251T=CGMCC 1.7012T)is galactosidase and arginine dihydrolase while negative for the type strain of the species Kosakonia oryzae and was lysine decarboxylase; positive for oxidation of arabinose, isolated from surface-sterilized roots of the wild rice spe- cellobiose, citrate, fructose, galactose, gluconate, glucose, cies Oryza latifolia grown in Guangdong, China [3]. glycerol, lactose, malate, maltose, mannitol, mannose, Here we present the summary of the features of the K. sorbitol, sucrose and trehalose (Table 1) [3, 8]. oryzae type strain Ola 51T and its complete genome se- The 16S rRNA gene sequence of K. oryzae Ola 51T quence, which provides a reference for resolving the was deposited in GenBank under the accession number phylogeny and taxonomy of closely related strains and EF488759 [3]. A phylogenetic analysis of the 16S rRNA gene sequences from the strains belonging to the genus Kosakonia and Escherichia coli ATCC11775T (the type * Correspondence: [email protected]; [email protected] strain of the type species of the type genus of the family T †Equal contributors Enterobacteriaceae) showed that K. oryzae Ola 51 is 3College of Agriculture, South China Agricultural University, Guangzhou most closely related to the strains belonging to the spe- 510642, China K. radicincitans – 1State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang cies (Fig. 2) [3, 8 11]. University, Hangzhou, China Full list of author information is available at the end of the article

© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Li et al. Standards in Genomic Sciences (2017) 12:28 Page 2 of 7

Table 1 Classification and general features of Kosakonia oryzae strain Ola 51T according to the MIGS recommendations [15] MIGS ID Property Term Evidence codea Classification Domain Bacteria TAS [34] Phylum Proteobacteria TAS [35] Class Gammaproteobacteria TAS [36, 37] Order “Enterobacteriales” TAS [38] Family Enterobacteriaceae TAS [39, 40] Genus Kosakonia TAS [8] Species Kosakonia oryzae TAS [3, 8] Type strain: Ola 51T TAS [3] Gram stain Negative TAS [3] Cell shape Rod TAS [3] Motility Motile TAS [3] Sporulation Non-sporulating TAS [3] Temperature 10–40 °C TAS [3] Fig. 1 Cell morphology of the Kosakonia oryzae type strain Ola 51T.The range bacterium was stained by uranyl acetate and observed by a transmission Optimum 28–37 °C TAS [3] electron microscope temperature pH range; 3.5–10; 6.0–8.0 TAS [3] Optimum Arabinose, cellobiose, Chemotaxonomic data Carbon source citrate, fructose, galactose, TAS [3, 8] Whole-cell fatty acids were extracted from cells grown gluconate, glucose, glycerol, aerobically at 28 °C for 24 h on the TSA medium lactose, malate, maltose, mannitol, mannose, sorbitol, according to the recommendations of the Microbial sucrose & trehalose Identification System (MIDI Inc., Delaware USA). The MIGS-6 Habitat Plants TAS [3] whole-cell fatty acid composition was determined using MIGS-6.3 Salinity 0 – 5% NaCl (w/v) TAS [3] a 6890 N gas chromatograph (Agilent Technologies, Santa Clara, USA) and the peaks of the profiles were MIGS-22 Oxygen Facultatively anaerobic TAS [3] requirement identified using the TSBA50 identification library version K. oryzae T MIGS-15 Biotic Free-living, endophytic TAS [3] 5.0 (MIDI). Ola 51 shows the typical cell fatty relationship acid profile of the genus Kosakonia [8]. The major fatty MIGS-14 Pathogenicity Not reported acids are C16:0,C18:1 ω7c,C16:1 ω7c/15:0 iso 2OH,C17:0 cyclo and C [8, 11]. MIGS-4 Geographic Guangzhou, Guangdong, TAS [3] 14:0 3OH/16:1 iso I location China MIGS-5 Sample September 12, 2005 TAS [3] Genome sequencing information collection Genome project history K. oryzae T MIGS-4.1 Latitude 23.1634171311 °N NAS Ola 51 was selected for sequencing based on its MIGS-4.2 taxonomic significance. The genome sequence is deposited Longitude 113.3534469581°E NAS in GenBank under the accession number CP014007. A MIGS-4.3 Depth 0.2 – 0.3 m below the TAS [3] summary of the genome sequencing project information surface and its association with MIGS version 2.0 [15] is shown in MIGS-4.4 Altitude 20 m NAS Table 2. a Evidence codes – IDA: Inferred from Direct Assay; TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Au- thor Statement (i.e., not directly observed for the living, isolated sample, but Growth conditions and genomic DNA preparation based on a generally accepted property for the species, or anecdotal evi- K. oryzae Ola 51T was grown aerobically in liquid Luria- dence). These evidence codes are from the Gene Ontology project [41] Bertani medium at 30 °C until early stationary phase. The genome DNA was extracted from the cells by using Genome sequencing and assembly a TIANamp bacterial DNA kit (Tiangen Biotech, Beijing, The genomic DNA of K. oryzae Ola 51T was con- China). DNA quality (OD260/OD280 = 1.8) and quantity structed into 8 – 11 kb insert libraries and sequenced (22 μg) were determined with a Nanodrop spectrometer using PacBio SMRT sequencing technology [16] at the (Thermo Scientific, Wilmington, USA). Duke University Genome Sequencing & Analysis Core Li et al. Standards in Genomic Sciences (2017) 12:28 Page 3 of 7

Fig. 2 Phylogenetic tree based on the 16S rRNA gene sequences showing the phylogenetic position of the Kosakonia oryzae type strain Ola 51T (●) and other strains belonging to the genus Kosakonia. The sequences were aligned using the SINA (SILVA Incremental Aligner) Alignment Service [42] and were constructed to the phylogenetic tree with the neighbor-joining algorithm and the Kimura 2-parameter model integrated in the MEGA 5.2 program [43]. Bootstrap values (>50%) of 1,000 tests are shown at the nodes. The tree was rooted on the outgroup Escherichia coli ATCC 11775T. The GenBank accession numbers of the sequences are indicated in brackets; * indicates the accession number of a contig of the whole genome sequence. The scale bar indicates 0.1% substitutions per site

Resource. Sequencing was run on two SMRT cells Genome annotation and resulted in 124,997 high-quality filtered reads Automated genome annotation was done using the with an average length of 8,260 bp. High-quality reads NCBI Prokaryotic Genome Annotation Pipeline [17]. were assembled by the RS_HGAP_Assembly.3 in the Functional annotations were done by searching against SMRT analysis v2.3.0. The final assembly produced the KEGG [18], InterPro [19], and COG [20] databases. 128-fold coverage of the genome. Genes with signal peptides were predicted using SignalP [21]. Genes with transmembrane helices were predicted using TMHMM [22].

Table 2 Genome sequencing project information for Kosakonia T Genome properties oryzae strain Ola 51 The genome of K. oryzae Ola 51T contains one circular MIGS ID Property Term chromosome (Fig. 3). The chromosome contains MIGS 31 Finishing quality Finished 5,303,342 nucleotides with 54.0% G + C content. The MIGS-28 Libraries used PacBio 8 –11 Kb library genome contains 4,926 predicted genes, 4773 protein- MIGS 29 Sequencing platforms PacBio RS II coding genes, 105 RNA genes (16 rRNA genes, 76 tRNA MIGS Fold coverage PacBio 128 × genes, and 13 ncRNA genes), 48 pseudo genes, and 1 31.2 CRISPR repeats. Among the 4,773 protein-coding genes, MIGS 30 Assemblers HGAP Assembly.3 in SMRT analysis- 3,765 genes (78.88%) have been assigned functions, while 2.3.0 1008 genes (21.12%) have been annotated as hypothet- MIGS 32 Gene calling method GeneMarkS+ ical or unknown proteins (Table 3). The distribution of Locus Tag AWR26 genes into COG functional categories is presented in Table 4 and Fig. 3. Genbank ID CP014007 GenBank Date of June 6, 2016 Insights from the genome sequence Release The genome sequences of K. cowanii JCM 10956T, GOLD ID Gp0154734 K. radicincitans DSM 16656T (=D5/23T) [23], K. BIOPROJECT PRJNA309028 radicincitans UMEnt01/12 [24], K. radicincitans YD4 MIGS 13 Source Material LMG 24251T = CGMCC 1.7012T [25], K. sacchari SP1T [26], “K. pseudosacchari” JM- Identifier 387T [11], K. oryzae KO348 [27], and Enterobacter Project relevance Taxonomy, agriculture, plant-microbe sp. R4-368 [28] which was close to K. sacchari SP1T interactions [26] had been deposited in the GenBank database. Li et al. Standards in Genomic Sciences (2017) 12:28 Page 4 of 7

Fig. 3 Circular map of the chromosome of the Kosakonia oryzae strain Ola 51T. From outside to the center: CDS on forward strand colored according to their COG categories (oranges/reds: information storage and processing; greens/yellows: cellular processes and signaling; blues/ purples: metabolism; grays: pooly characterized), CDS and RNA genes on forward strand, CDS and RNA genes on reverse strand, CDS on reverse strand colored according to their COG categories, GC content, and GC skew. The circular map was generated by CGView [44]

The genome ANIs (Additional file 1: Table S1) between Ola 51T and the other strains belonging to the genus Kosakonia were calculated using the Orthologous Average Nucleotide Identity tool [29]. Table 3 Genome statistics The cut-off ANI value for species boundary was set Attribute Value % of Total at 95% - 96% [30]. The ANI value (95.85%) between K. oryzae T K. radicincitans T Genome size (bp) 5,303,342 100 Ola 51 and DSM 16656 is in the fuzzy zone 95% - 96%. The digital DDH DNA coding (bp) 4,613,400 86.99 value between Ola 51T and DSM 16656T calculated DNA G + C (bp) 2,864,594 54.01 by the Genome-to-Genome Distance Calculator [31] DNA scaffolds 1 100 with the Formula 2 is 66.2%, below the 70% cut-off Total genes 4,926 100 value for species boundary. Moreover, Ola 51T and T Protein-coding genes 4,773 96.89 DSM 16656 were differentiated by metabolic phe- RNA genes 105 2.13 notypes [3, 11] and ribosomal protein mass profiles [5]. Therefore, K. oryzae and K. radicincitans are Pseudo genes 48 0.97 closely related sister species. Genes in internal clusters ND Strain YD4 was closer to K. radicincitans DSM 16656T Genes with function prediction 3765 76.43 than K. oryzae Ola 51T on the phylogenetic tree based on Genes assigned to COGs 4237 86.01 the 16S rRNA genes (Fig. 2). However, the ANI value and Genes with Pfam domains 4416 89.65 the digital DDH value between YD4 and K. radicincitans T Genes with signal peptides 432 8.77 DSM 16656 is 95.56% and 64.4%, respectively, while between YD4 and K. oryzae Ola 51T is 97.04% and 74.3%, Genes with transmembrane helices 1179 23.93 respectively. Therefore, the strain YD4 belongs to K. CRISPR repeats 1 0.02 oryzae but not K. radicincitans. Li et al. Standards in Genomic Sciences (2017) 12:28 Page 5 of 7

Table 4 Number of genes associated with general COG DotUandClpV)andsecretedproteins(VgrGandHcp)of functional categories thetypeVIsecretionsystem,whichmayplayaroleinthe Code Value %age Description plant-associated lifestyle [32]. Except K. radicincitans DSM T J 194 4.06 Translation, ribosomal structure and biogenesis 16656 and UMEnt01/12, these strains contain the most A 1 0.02 RNA processing and modification structural proteins (YscCJRSTUVN) of the type III secretion system, which is not widespread among the previously stud- K 414 8.67 Transcription ied endophytic bacteria [32]. L 140 2.93 Replication, recombination and repair These plant-associated Kosakonia strains contain genes B 0 0 Chromatin structure and dynamics contributing to multiple plant growth-promoting D 35 0.73 Cell cycle control, Cell division, chromosome activities. They all contain the nif gene cluster (nifJHDK- partitioning TYENXUSVWZMFLABQ) for the Mo-Fe nitrogenase- V 60 1.26 Defense mechanisms dependent nitrogen fixation, the genes encoding indole-3- T 278 5.82 Signal transduction mechanisms acetaldehyde dehydrogenase, aspartate aminotransferase, M 270 5.66 Cell wall/membrane biogenesis aromatic amino acid aminotransferase and phenylpyruvate N 163 3.42 Cell motility decarboxylase for producing the phytohormone auxin, and the budABC genes for producing volatile acetoin and U 123 2.58 Intracellular trafficking and secretion 2,3-butanediol which induce plant systemic resistance to O 154 3.23 Posttranslational modification, protein turnover, pathogens [33]. In addition, K. oryzae Ola 51T and YD4, chaperones and K. radicincitans DSM 16656T and UMEnt01/12 also C 287 6.01 Energy production and conversion contain the anf gene cluster (anfHDGK) for the Fe-Fe G 428 8.97 Carbohydrate transport and metabolism nitrogenase-dependent nitrogen fixation. In contrast, the E 476 9.97 Amino acid transport and metabolism clinical strain K. cowanii JCM 10956T does not contain F 93 1.95 Nucleotide transport and metabolism the nif gene cluster. H 188 3.94 Coenzyme transport and metabolism Conclusions I 152 3.18 Lipid transport and metabolism The phylogeny of the members of the genus Kosakonia P 293 6.14 Inorganic ion transport and metabolism based on the 16S rRNA gene sequences is roughly in Q 98 2.05 Secondary metabolites biosynthesis, transport agreement with their overall genome relatedness. The and catabolism complete genome sequence of K. oryzae Ola 51T pro- R 502 10.52 General function prediction only vides the reference genome for genomic identification of S 422 8.84 Function unknown strains belonging to K. oryzae. Analyses of the overall - 536 11.23 Not in COGs genome relatedness indices (ANI and digital DDH The total is based on the total number of protein coding genes in the genome values), easily and reliably show that K. oryzae and K. radicincitans are closely related sister species and that Strain KO348 was grouped with K. sacchari SP1T, the strain YD4, which shows close 16S rRNA gene-based Enterobacter sp. R4-368, and “K. pseudosacchari” JM- phylogeney to K. radicincitans and was classified into K. 387T on the phylogenetic tree based on the 16S radicincitans, belongs to K. oryzae. As well as YD4, rRNA genes (Fig. 2). The ANI value between KO348 which is able to promote growth of the yerba mate and K. oryzae Ola 51T is 84.04%. The strain KO348 plants in low-fertility soils [14], K. oryzae Ola 51T con- thus does not belong to K. oryzae.TheANIvaluebe- tains both the nif gene cluster and the anf gene cluster tween KO348 and Enterobacter sp. R4-368 [27], K. for nitrogen fixation and genes contributing to produc- sacchari SP1T,or“K. pseudosacchari” JM-387T is tion of auxin and volatile acetoin and 2,3-butanediol. 98.80%, 94.56%, or 94.05%, respectively. Therefore, Therefore, K. oryzae Ola 51T may be able to promote KO348 and R4-368 belong to the same species, likely plant growth. Genomic analyses also show that K. oryzae a novel species closely related to K. sacchari and “K. Ola 51T and YD4 may have the type III and VI secretion pseudosacchari”. systems and thus motivate us to study the functions of K. oryzae Ola 51T and YD4, K. radicincitans DSM 16656T the type III and VI secretion systems in the interactions and UMEnt01/12, K. sacchari SP1T, “K. pseudosacchari” JM- between beneficial Kosakonia bacteria and plants. 387T,andKosakonia sp. KO348 and R4-368 were all isolated from plants. Their genomes contain genes encoding multiple Additional file enzymes degrading plant cell wall polysaccharides and re- moving reactive oxygen species, likely facilitating endophytic Additional file 1: Table S1. Average nucleotide identities (ANIs) colonization [32]. They all contain genes encoding the regu- between genomes of the strains belonging to the genus Kosakonia. (DOC 38 kb) latory protein (Fha1) and structural proteins (Lip, IcmF, Li et al. Standards in Genomic Sciences (2017) 12:28 Page 6 of 7

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